Purification of rGFP: Lab Report for Experiment 4 in BIOL 3380

BIOL 3380 Experiment 4 - Lab Report Purification of rGFP using Ni +2 -agarose column Name: Circle your lab section: T–AM W-AM R-AM F-AM T–PM W-PM R-PM F-PM Instructor: Jing Pan Graduate TA: Date: 10/05/2023 Partner: 4-1

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Experiment 4 – Lab Report Purification of rGFP using Ni +2 -agarose column To save time, simply edit this word document by inserting your answers after each question/space. Please denote your answers by using bold , italic s, or colored font. Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate. 1. (4pts) The breaking buffer that we use this week contains 10mM Tris, pH 8.0, 150mM NaCl. The elution buffer is breaking buffer that also contains 300mM imidazole. The instructor made 3L of elution buffer. The instructor had 500ml of 1.5M of Tris (121.1 g/mole) (pH8.0), 750ml of 5M NaCl (MW = 58.44 g/mole) and 1.5L of 3M imidazole (68.08 g/mole). How much of each stock solution did they need? Show your calculations for credit. CONCENTRATION OF TRIS C1V1=C2V2 1 X V1 = 0.01M X 250 mL = 2.5 mL V1= 2.5 mL CONCENTRATION OF NaCl C1V1=C2V2 1 X V1 = 0.15 M X 250 mL = 7.5 ml V1= 7.5 mL CONCENTRATION OF IMIDAZOLE C1V1=C2V2 1 X V1 = 0.03 M X 250 mL = 75 mL. V1= 75 mL 2. (4pts) Based on the rGFP cloning procedure and GFP sequence described in lecture 3, construct a schematic diagram figure of rGFP. An example schematic was presented in lecture 4. Be sure to include important domain/region names, chromophore location, and amino acid distances. This figure can be hand drawn and inserted as a picture. 4-3

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3. (4pts) After you performed the freeze-thaw cycle to lyse the bacteria, you pelleted the debris using a centrifuge. You expect the supernatant to fluoresce, but the pellet also fluoresced. What are two reasons why this pellet fluoresced green? (Yes, I know it contained rGFP…. but why?) Since the freeze-thaw procedure is not completely effective, there are some unlysed cells present in the pellet. Due to the E. coli still containing the GFP protein, this would result in some GFP protein within cells being in the pellet rather than the supernatant. Also, the GFP (Green fluorescent protein) protein might still be present in the cellular debris, which could explain why the pellet fluoresced green. Since every piece of cellular debris descends to the pellet, some GFP would also stay there. 4. (3pts) W1 should not have fluoresced. Why? (I know it did not contain rGFP, but why?) W2-W4 should have fluoresced slightly. Give two possible reasons why. (Yes, I know it contained rGFP…. but why?) Fluorescence requires the fluorophore to absorb excitation energy, which causes it to emit light with a longer wavelength. Fluorescence wouldn't happen if the excitation energy wasn't present or wasn't enough in W1. W2-W4 exhibited slight fluorescence because of the potential presence of residual rGFP in the growth medium and the release of extracellular vesicles or membrane fragments during the sample processing steps. Photobleaching, which is the irreversible degradation of the fluorophore's ability to glow as a result of excessive light exposure, can affect GFP and other fluorescent proteins. The GFP molecules may have undergone photobleaching if W2-W4 was subjected to the strong or prolonged excitation light, which would have resulted in a decrease in fluorescence intensity. 5. (2pts) Looking at the qualitative rGFP fluorescence data, which wash fraction had the highest activity? Which elution fraction had the highest activity? Is this consistent with your raw quantitative fluorescence data (yes/no)? W2 and E2 had the highest activity, this was consistent with my raw quantitative data. 4-4

6. (3pts) Insert an image of your raw quantitative fluorescence data. Calculate the total amount of rGFP activity you have in the E2 fraction (this is the “scaled-up” data). Show your work. 7. (6pts) Construct an activity elution profile (an example was shown in lecture 4) using the scaled-up quantitative fluorescence values for W1-W6 and E1-E6 . Do NOT report the raw RFU data. Include an appropriate title and labeled/scaled axes. Do not include a figure caption. 4-5

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8. (2pts) Based upon the quantitative data, what two fractions would be the best to pool together for application to a subsequent column (like an ion exchange or gel filtration column)? E2 and E1 would be the best because they have the highest RFUS. 9. (2pts) A fellow student attempts to follow the protocol. They know that the GCE that was applied to the column was fluorescing, however fractions E1-E10 do not fluoresce. What two student errors could have occurred during the Ni +2 Agarose purification procedure to obtain this result? The student might have used breaking buffer instead of elution buffer, which would have prevented the GFP from eluting. The student might also not have allowed the GFP enough time to attach to the column before washing with breaking buffer, which would have resulted in the GFP washing out too soon. 4-7

Lab 4 Report F23

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